PRODUCT INFORMATION Chromalite resins for reverse-phase chromatography, adsorption and SPE

PRODUCT INFORMATION Chromalite resins for reverse-phase chromatography, adsorption and SPE Presents an overview of the advantages of Purolite s Chromalite polymeric resins over silica for the purification of biomolecules in reverse-phase chromatography.

Table of Contents Chromalite product line... 2 Reverse-phase chromatography... 3 Advantages of synthetic polymeric material over silica... 3 Chromalite polymers for reverse-phase chromatography... 3 Packaging options... 3 Different resins for different applications... 4 Chromalite AD... 5 Chromalite PCG... 5 Chromalite MN... 7 ph and chemical stability... 8 Column efficiency testing... 9 Packing procedures... 10 Mechanical stability/back pressure... 11 Applications... 12 Insulin... 12 APIs: Peptides obtained by fermentation... 16 Peptides... 17 Aromatic compounds... 19 Chromalite products... 21 HPLC columns... 22

Chromalite product line Mechanical robustness, inertness, ph stability, compatibility with both polar and non-polar organic solvents and even water are the most desirable properties of modern chromatographic resins. Purolite provides a comprehensive line of products for chromatographic applications, ranging from ion exchange resins to non-functionalized resins for reverse-phase chromatography (RPC), with particle size ranges from 10 micron to up to > 200 micron. Most products are characterized by narrow particle size distribution. Smaller particle sizes (3, 4 and 5 micron) are available on request for analytical HPLC applications. Table 1 shows the complete Chromalite product portfolio, including ion exchange resins (CGA, CGC, SBG, SBM products) and RPC resins. Chromalite ion exchange resins are described in detail in a separate technical brochure, Chromalite CGA and CGC Ion Exchange Resins, which can be found on www.purolite.com. This information guide looks specifically at Chromalite polymeric resins for reverse-phase chromatography (RPC). Table 1 Overview of Chromalite products by particle size and functional group TYPE CATION EXCHANGE ANION EXCHANGE REVERSE PHASE MEAN PARTICLE SIZE* 10 μm 15 μm 35 μm 50 μm 75 μm 125 μm > 200 μm 10AD1 10AD2 10MN 15SBG 15AD1 15AD2 15MN PCG1200F15 PCG600F PCG900F PCG1200F CGC50X2 CGC50X4 CGC50X8 CGA50X2 CGA50X4 CGA50X8 50SBM 70MN PCG600M PCG900M PCG1200M CGC100X2 CGC100X4 CGC100X8 CGA100X2 CGA100X4 CGC100X8 PCG600C PCG900C PCG1200C CGC200X2 CGC200X4 CGC200X8 CGA200X2 CGA200X4 CGA200X8 * Smaller particle sizes are available by request. Table 2 shows the range of applications of Chromalite RPC products depending on particle size. Table 2 Chromalite applications depending on resin particle size CHROMALITE SIZE RANGE (μm) 10, 15 APPLICATION Preparative RP-HPLC Process/polishing of small molecules and synthetic biomolecules 35 Process/polishing of synthetic biomolecules 50, 75 Low-pressure chromatography, front-end capture, desalting, and purification applications of small organic molecules and synthetic biomolecules (peptides and oligonucleotides) Solid phase extraction > 75 Large-scale purification of small organic molecules and synthetic biomolecules 2 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Reverse-phase chromatography Reverse-phase chromatography is a proven technique for the purification of biomolecules and results from the adsorption of molecules onto a hydrophobic solid support in a polar mobile phase. Decreasing the mobile phase polarity by adding more organic solvent reduces the hydrophobic interaction between the solute and the solid support resulting in desorption. The more hydrophobic the molecule, the more time it will spend on the solid support and the higher the concentration of organic solvent required to promote desorption. RPC applies to a very wide range of molecules including charged and polar molecules Variables such as organic solvent type and concentration, ph and temperature are used to optimize and control separation in RPC Once the stationary phase is packed in a column, RPC columns are robust, efficient and stable RPC media are robust and tolerate a wide range of ph, use of harsh chemicals, wide temperature range and are easily regenerated and cleaned At process scale, RPC is applied to organic compounds like APIs, biomolecules like peptides and oligonucleotides. RPC is rarely used for purification of larger biomolecules since the presence of the organic solvent can cause denaturation of proteins and destroy their biological activity. For purification of proteins, more hydrophilic matrices like agarose or acrylates are preferred. There are exceptions, such as the successful industrial purification by reverse phase of insulin, which will be described later. Reverse-phase chromatography is the most common analytical HPLC technique. There are many different stationary phases available for method optimization, like silica functionalized with hydrophobic groups (C18, C8 silica) or the more robust polymeric phases like Chromalite. Advantages of synthetic polymeric material over silica Polymeric resins are becoming used more frequently in modern liquid chromatography applications thanks to great improvements in resin design and manufacturing technology and the discovery of new applications. Modern polymers have overcome earlier problems associated with their use such as swelling. High rigidity and strength of new polymers now allow their use at high eluent flow rates resulting in faster analysis and increased column life. Improvements in synthetic resins have also increased efficiency, particularly in comparison to even the best silica materials. At times, use of polymeric materials can achieve efficiencies exceeding 100,000 plates/m. In addition, polymers offer distinct advantages over silica packing for particular applications. Silica media are sensitive to more extreme ph conditions, additives such as metals and buffer salts. In comparison, polymeric resins are particularly useful for purification and analysis of amino acids, peptides, small proteins such as insulin, organic acids, carbohydrates, and inorganic cations and anions. Chromalite polymers for reverse-phase chromatography Purolite offers a wide range of Chromalite products for reverse- phase chromatography with particle size ranging from 10 micron to greater than 200 micron. All these products are manufactured with the highest industry standard to achieve: high mechanical stability uniform particle size control of porosity high chemical stability More accurate separations Packaging options: Slurry: 20% ethanol for particle size lower than 45 micron (See Table 3) Wet: for all products with particle size higher than 45 micron (See Table 4) Dry: specific for resins used for solid phase extraction by reverse-phase mode (MN products) for easy cartridge filling and packing 3 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Table 3 Chromalite products with particle size < 45 micron (supplied as 20% EtOH slurry) PRODUCT PARTICLE SIZE * (90% in range) (μm) SURFACE AREA (m 2 /g) PORE DIAMETER (Å) BULK DENSITY (g dry /ml of bed volume) 10AD1 9 11 > 400 100 0.28 0.33 10AD2 9 11 > 400 300 700 0.28 0.33 15AD1 9 11 > 400 100 0.28 0.33 15AD2 13.5 16.5 > 400 300 700 0.28 0.33 10MN 9 11 > 1400 30 0.28 0.33 15MN 13.5 16.5 > 1400 30 0.28 0.33 PCG600F 20 55 > 700 100 0.20 0.24 PCG900F 20 55 > 600 200 300 0.17 0.20 PCG1200F15 12 18 > 600 300 700 0.16 0.19 PCG1200F 20 55 > 600 300 700 0.16 0.19 Smaller particle sizes are available by request. * 90% in the range Table 4 Purolite Chromalite resins with particle size >45 micron (supplied as wet) PRODUCT PARTICLE SIZE * (μm) SURFACE AREA (m 2 /g) Different resins for different applications PORE DIAMETER (Å) BULK DENSITY (g dry /ml of bed volume) 70MN 65 75 > 1400 30 0.28 0.33 <5 PCG600M 50 100 > 700 100 0.20 0.24 55 60 PCG600C 85 155 > 700 100 0.20 0.24 55 60 PCG900M 50 100 > 600 200 300 0.17 0.20 65 70 PCG900C 85 155 > 600 200 300 0.17 0.20 65 70 PCG1200M 50 100 > 600 300 700 0.16 0.19 65 70 PCG1200C 85 155 > 600 300 700 0.16 0.19 65 70 Supplied in dry form for easy packing of SPE cartridges. Available in wet form by request. * 90% in the range TOTAL MOISTURE AS SHIPPED (%) Chromalite AD and Chromalite PCG rigid polymeric reverse-phase resins are used extensively for analysis and purification in reverse-phase chromatography. The range of particle sizes provides scalability from an analytical platform through to industrial high-performance purification. Purolite offers three different resins featuring a 4 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

selection of particle sizes, porosities and surface areas, all producing stable packed columns with excellent flow rates, chemical stability and capacity. Chromalite AD Chromalite AD1 and AD2 are hydrophobic polystyrene porous adsorbents of high divinylbenzene content with outstanding chemical and physical stability. Chromalite AD matrices are designed for preparative reverse-phase chromatography with particle size of 10 or 15 micron. Chromalite AD1 and AD2 HPLC resins are inherently hydrophobic and an excellent alternative to C18 or C8 silica matrices. Chromalite AD1 has a medium porosity (100Å), ideal for separation of medium/small molecules with MW < 1000Da, and can substitute C8/C18 silica with porosity of 50 100Å. Chromalite AD2 has a higher porosity (300 700Å), which is ideal for separation of larger molecules (> 1000Da) such as synthetic oligomers, biomolecules, peptides, small proteins and oligonucleotides. Figure 1 Electron microscopy of Chromalite AD1 (15 micron) a) Uniform particle size distribution b) Controlled porosity Chromalite PCG Chromalite PCG resins are macroporous hydrophobic polystyrene adsorbents intended for use in high-, medium- and low-pressure reverse-phase chromatography. Three different porosities enable Chromalite PCG resins to accommodate a variety of molecule sizes. High porosity: Chromalite PCG1200 with a porosity of 300 700 Å Medium porosity: Chromalite PCG900 with a porosity of 200 300 Å Low porosity: Chromalite PCG600 with a porosity of 100 Å Chromalite PCG resins can be used for RP-HPLC or medium- and low-pressure RP-LC due to the particle sizes offered: 12 18 micron (Chromalite PCG1200F15) 20 55 micron (F grade) 50 100 micron (M grade) 85 150 micron (C grade) 5 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

PUROLITE PRODUCT INFORMATION Figure 2 Electron microscopy of Chromalite PCG1200F15 (12 18 micron) a) Uniform particle size distribution b) Controlled porosity Figure 3 Optical photographs of PCG1200F (20 55 micron) and PCG1200M (50 100 micron) a) F grade resin 6 b) M grade resin Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Chromalite MN Principally differing from conventional styrene-divinylbenzene copolymers like Chromalite PCG and AD, Chromalite MN, hypercrosslinked polystyrene is obtained by an extensive post-crosslinking of long polystyrene chains. The resulting structure of hypercrosslinked polystyrene is an expanded, rigid three-dimensional network. Chromalite MN takes advantage of Purolite proprietary technology. 1 The open-work-type hypercrosslinked network displays an extremely high apparent inner surface area (up to 1000-1500 m 2 /g) and no change in solvent uptake in both polar and non-polar resins, which enables good compatibility of the material with all mobile phases from hexane to methanol and water. The whole interior of the hypercrosslinked polystyrene bead is well accessible to small/medium size analytes thanks to the network composition formed by small micro pores of about 2.0 to 4.0 nm in diameter. Solid phase extraction (SPE) is an increasingly useful sample preparation technique. With SPE, many of the problems associated with liquid/liquid extraction can be prevented, such as incomplete phase separations, less-than-quantitative recoveries, use of expensive, breakable specialty glassware and disposal of large quantities of organic solvents. SPE is more efficient than liquid/liquid extraction, yields quantitative extractions that are easy to perform, is rapid and can be automated. It also reduces solvent use and lab time. Chromalite MN matrices are ideal for solid-phase extraction (SPE) of small and medium organic compounds with aromatic functions. The robustness of Chromalite MN resins also makes this hypercrosslinked non-modified polystyrene resin ideal for HPLC applications. Due to compatibility with any type of organic solvents and water, Chromalite MN can operate throughout a wide range of eluent polarity. Combining the non-polar Chromalite MN with an aqueous or polar aqueous-organic mobile phase corresponds to conventional reverse-phase mode of chromatography. While hypercrosslinked polystyrene is chemically stable and easily withstands temperatures of 200 C and higher, the target application of Chromalite MN is modern high-temperature HPLC. Enhanced temperatures, through facilitating mass transfer, substantially increase column efficiency and reduce analysis time. Chromalite MN ensures the following: Exceptional lot-to-lot reproducibility High recoveries and yields resulting from exceptional high surface area/binding sites Exceptional ph stability (1 to 14) Excellent packing and storage qualities due to the dry supply of the resin Chromalite MN is manufactured with very narrow particle distribution and it is offered in three different particle sizes: 9 11 micron: Chromalite 10MN 13.5 16.5 micron: Chromalite 15MN 65 75 micron: Chromalite 70MN Small particle sizes (5 micron) are also available by request. 1 [1] C.S. Sychov, M.M. Ilyin, V.A. Davankov, K.O. Sochilina. Elucidation of retention mechanisms on hypercrosslinked polystyrene used as column packing material for high performance liquid chromatography. / J. Chromatogr. A 1030 (2004) 17-24 [2] V.A. Davankov, C.S. Sychov, M.M. Ilyin, K.O. Sochilina. Hypercrosslinked polystyrene as a novel type of high-performance liquid chromatography column packing material. Mechanisms of retention. / J. Chromatogr. A 987 (2003) 67-75 [3] V. Davankov, M. Tsyurupa, M. Ilyin, L. Pavlova. Hypercross-linked polystyrene and its potentials for liquid chromatography: a mini-review, / J. Chromatogr. A 965 (2002) 65 7 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Resins are offered in dry form for the analysis of biological fluids to make packing of SPE cartridges easy and reproducible. Figure 4 Optical photographs of Chromalite 70MN ph and chemical stability Chromalite AD, PCG and MN are poly(styrene/divinylbenzene) copolymers with excellent chemical stability. This gives flexibility in the choice of elution conditions and cleaning regimes, including the use of sodium hydroxide, which increases sample throughput and the number of cycles achieved per column. All resins are stable from ph 1 to 14. 8 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Column efficiency testing The preparation and qualification of packed columns are important steps to ensure robustness and safety for both the purification process and the final product. Column efficiency testing plays a central role in the qualification and monitoring of packed bed performance. Column packing efficiency tests were performed using sodium chloride as a tracer. 0.4 M sodium chloride was added to the eluent to suppress any possible charge interaction effects between the resin and a tracer. Column packing efficiency test was evaluated based on HETP and asymmetry factor. Evaluation tests were carried out as follows: Table 5 Column efficiency testing for Chromalite PCG600, Chromalite PCG900 and Chromalite PCG1200 with NaCl tracer GRADE PARTICLE SIZE (μm) PLATES (per meter) PLATE HEIGHT (cm) ASYMMETRY* F 20 55 > 3000 0.03 1.4 M 50 100 > 1800 0.04 1.8 C 85 155 > 900 0.08 0.9 * An asymmetry factor close to AS = 1 is ideal. A typical acceptable range would be 0.8 1.8. Experimental conditions: Column type: HiScale 16/20 Bed height: 20 cm Bed volume: 40 ml Flow rate: 200 cm/h Equilibration: 2.5 CV 0.4 M NaCl Tracer: 0.01 CV 2 M NaCl Detection: Conductivity, ms/cm Elution: 1.5 CV 0.4 M NaCl System: ÄKTA avant 25 9 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Table 6 Column efficiency testing for Chromalite PCG600, Chromalite PCG900 and Chromalite PCG1200 with Lysozyme tracer GRADE PARTICLE SIZE (μm) PLATES (per meter) PLATE HEIGHT (cm) ASYMMETRY* F 20 55 > 1800 0.05 2.5 M 50 100 > 500 0.16 1.6 C 85 155 > 400 0.23 1.2 * An asymmetry factor close to AS = 1 is ideal. A typical acceptable range would be 0.8 1.8. Experimental conditions: Column type: HiScale 16/20 Bed height: 20 cm Bed volume: 40 ml Flow rate: 300 cm/h Equilibration: 1 CV 20% ethanol in water Tracer: 0.01 CV of 1% acetone in 20% ethanol solution Detection: UV1, 280 nm Elution: 2.5 CV of 20% ethanol in water System: ÄKTA avant 25 Quality, reproducibility and scalability Chromalite AD, PCG and MN resins are produced with the highest level of quality. The flexibility of particle size allows for applications to range from analytical/prep to process development. Packing procedures - Drain the resin of water/ethanol (EtOH) and replace with EtOH or methanol (MeOH). Avoid drying the resins. In case of supplied dried resins, these can be used directly to fill cartridges. - Suspension slurry should have a concentration of 5 20% by volume of adsorbent. It should be homogeneous with no aggregates. Mild treatment with ultrasound can be used to prevent aggregation. - To determine quantity for packing, see bulk densities within Tables 3 and 4. - Use 10 15% excesses of resin to fill columns and to allow some resin bed compression. - Pack preparative column at 50 bars pressure for the best performance. Analytical columns can be packed at 100 200 bar. Chromalite AD resins do not get damaged at pressures up to 300 bar, but can develop pore compression, which can deteriorate resin performance. Always keep packed column wet in alcohol/water eluent. To regenerate the resins, use 1 2 N caustic and 6 M thiourea, if needed. Also hydrochloric and acetic acids in any concentration could be applied to Chromalite. Avoid using pure toluene, chlorinated hydrocarbons, acetonitrile and acetone for column washing as well as any oxidizing substances. 10 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Mechanical stability/back pressure The pressure of a chromatography system is measured after the system pumps. This pressure is called system back pressure. If a column is attached to the system, the additional pressure is called column back pressure. Chromalite PCG and AD are fully stable to typical back pressures generated in chromatographic applications due to high cross-linking. Table 7 Typical pressure limits for Chromalite resins GRADE MAX BACK PRESSURE (bar) F15* 200 F 50 M 10 C 10 * 12 18μm particle size Figure 5 shows the excellent mechanical stability of Chromalite PCG1200F15 with particle size 12 18 micron under pressure. The resin is fully stable up to 200 bar before starting to show the typical profile of irreversible deformation. Compared to other commercially available resins with similar particle size, Chromalite PCG1200F15 shows outstanding performance. Figure 5 Pressure flow rate curve for Chromalite PCG1200 15 micron in a 4.0 ml (250 mm 4.6 mm) column The column was pre-equilibrated with water/i-propanol and back pressure was registered by increasing flow rate using i-propanol as eluent. Back pressure linearly increases up to a flow rate of 5 ml/min (1800 cm/hour) linear velocity. 11 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Applications Insulin Insulin is a widely used peptide hormone for the treatment of diabetes mellitus. It is composed of two chains of 21 amino acids and 30 amino acids held together by two disulfide bonds. An additional disulfide bond is formed within the short chain. Insulin has a molecular weight of about 5,800 Daltons. Nowadays, insulin is mainly produced using fermentation of recombinant micro organisms (Escherichia coli).the E. coli have had a human gene spliced into their DNA compelling them to produce human proinsulin. The pro-insulin is harvested by lysing the bacteria followed by clarification of the fermentation broth by centrifugation and filtration. The proinsulin has then to be refolded into its active tertiary structure by treatment in a refolding vessel with buffers of various concentrations. After enzymatic cleavage of proinsulin to obtain insulin, industrial purification of crude insulin proceeds via 1) ion exchange chromatography for the removal of impurities from fermentation and 2) reverse-phase chromatography for the removal of analogues of insulin. Figure 6 Insulin structure showing short and long chains The reverse-phase chromatography step Insulin-like components (desamido-, carbamoyl-, formyl-insulin and other forms) represent 6 to 20% of the fermentation mixture and are the most difficult molecules to be separated. Desamido-insulin is a key impurity in insulin purification. Its purification is particularly difficult for separation as it differs from insulin only in 1 amino acid moiety. This purification is perfomed using reversed-phase chromatography. Desamido-insulin can be efficiently separated from insulin with Chromalite PCG1200F15 (15 micron). Insulin resolution Mixtures of 85% insulin/10% of desamido-insulin/5% of other insulin-like impurities can be efficiently purified using Chromalite PCG1200F15 obtaining yields higher than 87% and purity higher than 98.4%. 12 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Scale-up purification Chromalite PCG1200 resins also allow efficient separation on the preparative scale. By keeping the same linear flow rate, same load per column volume and bed height, scale-up is easy and highly predictable. Chromalite PCG1200 resins allow the packing of columns in dimensions that suit optimal resolution and capacity needs. Figure 7 Performance comparison between PCG1200F15 15 m and C8 Silica (15 m) PCG1200F15 Column: 25x250 mm column Adsorbent: PCG1200F15 (15 μm) Sample: 10 mg mixture of 85% insulin / 10% of desamido-insulin / 5% of other insulin-like impurities insulin per 1 ml of resin Buffer A: 0.1 M acetic acid / 10% ethanol in water Buffer B: 50% ethanol, water Flow rate: 5.5 ml/min (70 cm/hour linear velocity) Elution: Gradient elution System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 280 nm C8 Silica Column: 25x250 mm column Adsorbent: C8 Silica (15 μm) Sample: 10 mg mixture of 85% insulin / 10% of desamido-insulin / 5% of other insulin-like impurities insulin per 1 ml of resin Buffer A: 0.1 M acetic acid / 10% ethanol in water Buffer B: 50% ethanol, water Flow rate: 5.5 ml/min (70 cm/hour linear velocity) Elution: Gradient elution System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 280 nm Advantages of Chromalite resins: PCG1200F15 offers the same resolutions as silica material with the advantages of synthetic materials such as mechanical robustness and the ability to use in all range of ph conditions. 13 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Figure 8 shows retained resolution upon scale-up with PCG1200 with different particle sizes. 2 Figure 8 - Retained resolution upon scale-up with PCG1200F15 and PCG1200F PCG1200F15 Column: Adsorbent: 25x250 mm column PCG1200F15 (12 18 μm) Sample: 10 mg mixture of 85% insulin / 10% of desamido-insulin / 5% of other insulin-like impurities insulin per 1 ml of resin Buffer A: 0.1 M acetic acid / 10% ethanol in water Buffer B: 50% ethanol, water Flow rate: 5.5 ml/min (70 cm/hour linear velocity) Elution: Gradient elution System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 280 nm PCG1200F Column: 25x250 mm column Adsorbent: PCG1200F (20 55 μm) Sample: 10 mg mixture of 85% insulin / 10% of desamido-insulin / 5% of other insulin-like impurities insulin per 1 ml of resin Buffer A: 0.1 M acetic acid / 10% ethanol in water Buffer B: 50% ethanol, water Flow rate: 5.5 ml/min (70 cm/hour linear velocity) Elution: Gradient elution System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 280 nm Advantages of Chromalite resins: Scaling up from PCG1200F15 to PCG1200F guarantees the same separation profile. Scaling up from laboratory to production scale is reproducible and predictable. 2 Tested by Institute of Bioorganic Chemistry, Experimental Biotech Plant, Russian Academy of Sciences, Laboratory of Proteins Isolation and Purification, Moscow. 14 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Figure 9 - Comparison with other synthetic resin CHP 55A 18 μm Column: 25x250 mm column Adsorbent: CHP 55A (18 μm) (Mitsubishi) Sample: 10 mg mixture of 85% insulin / 10% of desamido-insulin / 5% of other insulin-like impurities insulin per 1 ml of resin Buffer A: 0.1 M acetic acid / 10% ethanol in water Buffer B: 50% ethanol, water Flow rate: 5.5 ml/min Elution: Gradient elution System: Semi-preparative HPLC system ÄKTA Basic 100 (GE Healthcare, Sweden) Detection: UV-1 monitor, 280 nm Amberchrom HPR10 10 μm Column: 25x250 mm column Adsorbent: Amberchrom HPR10 (10 μm) (Dow) Sample: 10 mg mixture of 85% insulin / 10% of desamido-insulin / 5% of other insulin-like impurities insulin per 1 ml of resin Buffer A: 0.1 M acetic acid / 10% ethanol in water Buffer B: 50% ethanol, water Flow rate: 5.5 ml/min Elution: Gradient elution System: Semi-preparative HPLC system ÄKTA Basic 100 (GE Healthcare, Sweden) Detection: UV-1 monitor, 280 nm YMCbasic Silica C8 15 μm Column: 25x250 mm column Adsorbent: YMCbasic Silica C8 (15 μm) (YMC Co.) Sample: 10 mg mixture of 85% insulin / 10% of desamido-insulin / 5% of other insulin-like impurities insulin per 1 ml of resin Buffer A: 0.1 M acetic acid / 10% ethanol in water Buffer B: 50% ethanol, water Flow rate: 5.5 ml/min Elution: Gradient elution System: Semi-preparative HPLC system ÄKTA Basic 100 (GE Healthcare, Sweden) Detection: UV-1 monitor, 280 nm Advantages of Chromalite resins: Chromalite PCG1200F15 (see Figure 8) provides better resolution and performance than other commercially available polymeric resins or silica materials. Resolution obtained with PCG1200F15 is even better than using small-particle polymeric materials (10 micron) such as Amberchrom. 15 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

APIs: Peptides obtained by fermentation Bradykinin is a 9 amino acid peptide chain with a MW of 1059 that is used to decrease blood pressure. Bacitracin is a mixture of related cyclic oligopeptides (MW 1422 ) produced by microorganisms of the Bacillus species. Bacitracin is an effective topical antibacterial. Gramicidin is a heterogeneous mixture of six antibiotic compounds, gramicidins A, B and C, making up 80%, 6%, and 14% respectively. Gramicidins are oligopeptides containing 15 amino acids with MW of about 1880. Its therapeutic use is limited to topical antibacterial applications. Figure 10 Separation of a bradykinin (1), bacitracin (2) and gramicidin C (3) mixture using Chromalite PCG900F and Chromalite PCG600F PCG900F 20 55 μm Column: Adsorbent: Sample: Buffer A: Buffer B: Flow rate: Elution: System: Detection: 25x250 mm column PCG900F (20 55 μm) Mixture of brandykinin, bacitracin and gramicidin C 10 mm citric acid / 10% ethanol / water for injections 50% ethanol; water 7.5 ml/min Gradient elution Semi-preparative HPLC system ÄKTA UV-1 monitor, 220 nm PCG600F 20 55 μm Column: Adsorbent: Sample: Buffer A: Buffer B: Flow rate: Elution: System: Detection: 25x250 mm column PCG600F (20 55 μm) Mixture of brandykinin, bacitracin and gramicidin C 10 mm citric acid / 10% ethanol / water for injections 50% ethanol; water 7.5 ml/min Gradient elution Semi-preparative HPLC system ÄKTA UV-1 monitor, 220 nm Advantages of Chromalite resins: Chromalite PCG600F and Chromalite PCG900F are ideal for separation of small biomolecules such as small peptides as the polymeric structure of PCG resins allows for perfect separation of peptides. 16 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Peptides The capability of Chromalite resins is demonstrated in the resolution of peptide mixtures. Figure 11 Peptide separation using Chromalite resins and comparison with silica media 10AD2 10 μm Column: Adsorbent: 4.6x150 mm column 10AD2 (10 μm) Sample: Mixture of octapeptide (main peak at 16 minutes) and impurity (heptapeptide, minor peak at 11.5 minutes) Buffer A: 0.5% TFA in water Buffer B: Acetonitrile Flow rate: 1 ml/min Elution: Linear gradient 10 40% System: Waters Alliance HPLC Detection: UV-1 monitor, 220 nm Rs: 2.8 PLRP-S 300A 10 μm Column: Adsorbent: 4.6x150 mm column PLRP-S 300A (10 μm) Sample: Mixture of octapeptide (main peak at 16 minutes) and impurity (heptapeptide, minor peak at 11.5 minutes) Buffer A: 0.5% TFA in water Buffer B: Acetonitrile Flow rate: 1 ml/min Elution: Linear gradient 10 40% System: Waters Alliance HPLC Detection: UV-1 monitor, 220 nm Rs 2.1 Waters Symmetry Silica C18 5 μm Column: 4.6x150 mm column Adsorbent: Waters Symmetry Silica C18 (5 μm) Sample: Mixture of octapeptide (main peak at 16 minutes) and impurity (heptapeptide, minor peak at 11.5 minutes) Buffer A: 0.5% TFA in water Buffer B: Acetonitrile Flow rate: 1 ml/min Elution: Linear gradient 10 40% System: Waters Alliance HPLC Detection: UV-1 monitor, 220 nm Rs: 2.9 Advantages of Chromalite resins: Figures 11 and 12 show better separation efficiency (resolution Rs) of Chromalite resins compared to other commercial polymers and silica materials. 17 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Figure 12 Peptide separation comparison using Chromalite resins and a synthetic commercial resin PLRP-S 300A 10 μm Column: Adsorbent: Sample: Buffer A: Buffer B: Flow rate: Elution: System: Detection: 4.6x150 mm column PLRP-S 300A (10 μm) Mixture of octapeptide (main peak) and impurity (heptapeptide, minor peak) 0.5% TFA in water 0.5% TFA in 80% acetonitrile 1 ml/min Isocratic at 27% B Semi-preparative HPLC system ÄKTA UV-1 monitor, 220 nm PCG1200F15 15 μm Column: Adsorbent: Sample: Buffer A: Buffer B: Flow rate: Elution: System: Detection: 4.6x150 mm column PCG1200F15 (12 18 μm) Mixture of octapeptide (main peak) and impurity (heptapeptide, minor peak) 0.5% TFA in water 0.5% TFA in 80% acetonitrile 1 ml/min Isocratic at 27% B Semi-preparative HPLC system ÄKTA UV-1 monitor, 220 nm 10AD1 10 μm Column: Adsorbent: Sample: Buffer A: Buffer B: Flow rate: Elution: System: Detection: 4.6x150 mm column 10AD1 (10 μm) Mixture of octapeptide (main peak) and impurity (heptapeptide, minor peak) 0.5% TFA in water 0.5% TFA in 80% acetonitrile 1 ml/min Isocratic at 27% B Semi-preparative HPLC system ÄKTA UV-1 monitor, 220 nm Advantages of Chromalite resins: Figure 12 highlights that Chromalite 10AD1 can resolve as good as silica materials at 5 μm, which can present significant advantages in terms of scale-up and back pressure limitations. 18 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Aromatic compounds Figure 13 Separation of aromatic compounds using Chromalite resins and a commercial synthetic resin PLRP-S 300 A 10 μm Column: 4.6x150 mm column Adsorbent: PLRP-S 300 A (10 μm) Sample: Mixture of phenol (1)./.acetophenol (2) / nitrobenzene (3) / benzene (4) / toluene (5) Buffer A: Water Buffer B: Acetonitrile Flow rate: 1 ml/min Elution: Isocratic, 60% B System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 220 nm 10AD2 10 μm Column: 4.6x150 mm column Adsorbent: 10AD2 (10 μm) Sample: Mixture of phenol (1) / acetophenol (2) / nitrobenzene (3) / benzene (4) / toluene (5) Buffer A: Water Buffer B: Acetonitrile Flow rate: 1 ml/min Elution: Isocratic, 60% B System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 220 nm PCG1200F15 15 μm Column: 4.6x150 mm column Adsorbent: PCG1200F15 (15 μm) Sample: Mixture of phenol (1) / acetophenol (2) / nitrobenzene (3) / benzene (4) / toluene (5) Buffer A: Water Buffer B: Acetonitrile Flow rate: 1 ml/min Elution: Isocratic at 60% B System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 220 nm Advantages of Chromalite resins: The hydrophobic matrix of Chromalite resins for RPC optimizes separation of aromatic compounds and obtains the same efficiency as other commercially available resins. 19 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Figure 14 Separation of aromatic compounds using Chromalite 10MN 10μm and different elution conditions Column: 4.6x250 mm column Adsorbent: 10MN (10 μm) Sample: Model mix of benzaldehyde (1) / anisole (2) / cumene (3) / bromobenzene (4) / naphthalene (5) / anthraquinone (6) / anthracene (7) Buffer A: Water Buffer B: ACN/iPrOH 85/15 Flow rate: 1 ml/min Elution: Isocratic, 5% A System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 254 nm Column: Adsorbent: 4.6x250 mm column 10MN (10 μm) Sample: Model mix of benzaldehyde (1) / anisole (2) / cumene (3) / bromobenzene (4) / naphthalene (5) / anthraquinone (6) / anthracene (7) Buffer A: Chloroform Buffer B: Methanol Flow rate: 1 ml/min Elution: Isocratic, 50% A System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 254 nm Column: 4.6x250 mm column Adsorbent: 10MN (10 μm) Sample: Model mix of benzaldehyde (1) / anisole (2) / cumene (3) / bromobenzene (4) / naphthalene (5) / anthraquinone (6) / anthracene (7) Buffer A: Hexane Buffer B: Chloroform Flow rate: 1 ml/min Elution: Isocratic, 80% A System: Semi-preparative HPLC system ÄKTA Detection: UV-1 monitor, 254 nm Advantages of Chromalite resins: The polymer backbone of Chromalite RPC resin is stable to many organic solvents and offers incredible flexibility in separation conditions. It is possible to use organic solvents ranging from methanol, i-proh, ACN, hexane, chloroform, etc. The elution profile of the organic solvent changes the significantly, providing many possibilities for optimal resolution of target molecules. 20 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

Chromalite products Table 8 Chromalite resins for reverse-phase chromatography, adsorption and SPE PRODUCT CODE PRODUCT PARTICLE SIZE (90% in range) (μm) PACKING 06845-190 10AD1 9 11 10 g dry 06845-187 50 g dry 06845-193 100 g dry 06840-190 06840-187 06840-193 06895-190 06895-187 06895-193 06890-190 06890-187 06890-193 04685-190 04685-187 04685-193 04690-190 04690-187 04690-193 06910-190 06910-187 06910-193 04740-190 04740-187 04740-193 04750-186 04750-192 04750-195 04760-186 04760-192 04760-195 04770-190 04770-187 04770-193 04770-186 04770-192 04770-195 04790-186 04790-192 04790-195 06715-190 06715-187 06715-193 06720-190 06720-187 06720-193 06725-186 06725-192 06725-195 06730-186 06730-192 06730-195 10AD2 9 11 10 g dry 50 g dry 100 g dry 15AD1 13.5 16.5 10 g dry 50 g dry 100 g dry 15AD2 13.5 16.5 10 g dry 50 g dry 100 g dry 10MN 9 11 10 g dry 100 g dry 250 g dry 15MN 13.5 16.5 10 g dry 100 g dry 250 g dry 70MN 65 75 10 g dry 100 g dry 250 g dry PCG600F 20 55 10 g dry 50 g dry 100 g dry PCG600M 58 92 50 g wet 250 g wet 500 g wet PCG600C 100 140 50 g wet 250 g wet 500 g wet PCG900F 20 55 10 g dry 50 g dry 100 g dry PCG900M 58 92 50 g wet 250 g wet 500 g wet PCG900C 100 140 50 g wet 250 g wet 500 g wet PCG1200F15 12 18 10 g dry 50 g dry 100 g dry PCG1200F 20 55 10 g dry 50 g dry 100 g dry PCG1200M 58 92 50 g wet 250 g wet 500 g wet PCG1200C 85 155 50 g wet 250 g wet 500 g wet SUPPLIED AS Supplied as 20% EtOH slurry Supplied as 20% EtOH slurry Supplied as 20% EtOH slurry Supplied as 20% EtOH slurry Dry Dry Dry Supplied as 20% EtOH slurry Wet Wet Supplied as 20% EtOH slurry Wet Wet Supplied as 20% EtOH slurry Supplied as 20% EtOH slurry Wet Wet 21 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

HPLC columns HPLC columns are essential for optimal resolution in laboratory analysis. Purolite offers 150x4.6 mm pre-packed columns filled with Chromalite resins for reverse-phase chromatography. Columns are available with particle sizes averaging 10 micron and 15 micron, as shown in Table 9. Table 9 Analytical HPLC columns PRODUCT CODE CHROMALITE PARTICLE SIZE (μm) COLUMN SIZE 06845-601 10AD1 9 11 150 x 4.6 06840-601 10AD2 9 11 150 x 4.6 06895-601 15AD1 13.5 16.5 150 x 4.6 06890-601 15AD2 13.5 16.5 150 x 4.6 06715-601 PCG1200F15 13.5 16.5 150 x 4.6 The correct use of an HPLC column is critical for the life of the column and the accuracy of HPLC analysis. HPLC columns packed with Chromalite resins can be cleaned, regenerated and used following the same precautions used for silica-based HPLC columns, taking into consideration that a ph of 1 to 14 is acceptable. Figure 15 HPLC Column pre-packed with Chromalite resin 22 Chromalite for Reverse-Phase Chromatography, Adsorption and SPE 02 2014

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